Identification of small RNAs During the past ten years, we have carried out several different systematic screens for small regulatory RNA genes in E. coli. These screens, which have included computational screens for conservation of intergenic regions and direct detection after size selection or co-immunoprecipitation with the RNA binding protein Hfq, are all applicable to other organisms. We are now examining small RNA expression using tiled microarrays together with deep sequencing to further extend our identification of small RNAs, particularly antisense RNAs. Characterization of specific small RNAs A large focus of the group has been to elucidate the functions of the small RNAs in E. coli. Early on we showed that the OxyS RNA, whose expression is induced in response to oxidative stress, acts to repress translation through limited base pairing with target mRNAs. We also discovered that the OxyS action is dependent on the Sm-like Hfq protein, which functions as a chaperone to facilitate OxyS RNA base pairing with its target mRNAs. It is now clear that Hfq-binding small RNAs, which act through limited base pairing are integral to many different stress responses in E. coli. We found that the Hfq-binding RNA MicC, whose expression is induced in minimal medium and at low temperature, represses translation of the OmpC outer membrane porin. We also recently reported that the Hfq-binding small RNA FnrS, whose expression is induced by FNR upon a shift from aerobic to anaerobic conditions, acts to down regulate the levels of a variety of mRNAs encoding metabolic enzymes. These results suggest that the FnrS RNA extends the FNR regulon and increases the efficiency of anaerobic metabolism by repressing the synthesis of enzymes that are not needed under these conditions. In addition to small RNAs that act via limited base pairing, we have become interested in small antisense RNAs that have the potential to form extensive base pairing interactions with their mRNA targets encoded on the opposite strand. We previously showed that base pairing between the GadY RNA and the 3-untranslated region of the gadX mRNA encoded opposite GadY leads to increased levels of the gadX mRNA and GadX protein. Recently, we demonstrated that gadX is transcribed in an operon with gadW and that base pairing of GadY with the gadXW mRNA results in processing giving rise to two halves that accumulate to higher levels than the full length mRNA. Interestingly, multiple enzymes, including the double strand RNA-specific endoribonuclease RNase III, are involved in the GadY-direct cleavage. We have also found that a large class of antisense RNAs acts to repress the synthesis of small toxic proteins. For example, SymR represses the sense symE gene, which encodes a protein with properties of a toxin despite having homology to the MazE antitoxin. The importance of repressing symE expression is emphasized by the observation that the synthesis of the SymE protein is repressed by the LexA repressor, the SymR RNA and the Lon protease. In another study, we characterized the Sib RNAs, which are encoded by five repeats in E. coli K-12. Curiously, a phenotype reminiscent of plasmid addiction was observed for overexpression of the Sib RNAs, and further examination of the SIB repeat sequences revealed conserved open reading frames encoding highly hydrophobic 18-19 amino acid proteins (Ibs) opposite each sib gene. The Ibs proteins were found to be toxic when overexpressed, and this toxicity could be prevented by co-expression of the corresponding Sib RNA. Two other RNAs encoded divergently in another intergenic region were similarly found to encode a small hydrophobic protein (ShoB) and an antisense RNA regulator (OhsC). Most recently, computational screens together with experimental validation have shown that these small hydrophobic protein-antisense RNA gene modules, termed type 1 toxin-antitoxin modules, are much more widely distributed among bacteria than previously appreciated. Studies to further characterize other Hfq-binding RNAs and antisense RNAs and to elucidate the roles of small RNAs that act in ways other than base pairing are ongoing.